Membrane surface activity of Arf1 and coatomer. (A) Membrane surface activity of Arf1 analyzed in GUVs. GUVs containing 0.5 mol% 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-lissamine rhodamine B sulfonyl were incubated with 3.5 µM Arf1-wt and 0.4 µM ARNO in the presence or absence of 1 mM GTP as indicated and recorded in a confocal laser-scanning microscope (LSM 510; Carl Zeiss) with a 63× objective lens and a pinhole size equivalent to one Airy disk diameter. (B) Membrane surface activity of Arf1 and coatomer. Lipids containing the p23 lipopeptide were spotted on a glass surface and hydrated with buffer containing GTP and 50 nM of the exchange factor ARNO. The lipid surface was observed before (left) and after addition of 1 µM myristoylated Arf1-GDP (top right) or by coinjection of 1 µM Arf1 and 0.25 µM coatomer (cm; bottom right). (C) Dissecting membrane surface activities of Arf1 and coatomer. (a) Lipids containing the p23 lipopeptide were spotted on a glass surface and hydrated with buffer containing GTP and the exchange factor ARNO as described in this legend. The image was taken, and thereafter, Arf1 was added, and tubule formation was observed (Video 1). (b) 0.25 µM coatomer was added to the chamber, leading to an immediate and nonspecific loss of most tubular structures caused by capillary flow forces. One frame afterward (t = 0), remaining Arf1-generated tubules are depicted (arrows). (c–f) Images were taken at 2, 4, 6, and 8 s after the addition of coatomer. The rapid degradation of Arf1-generated tubules (arrows) was followed over time, whereas new tubular structures with a distinct morphology were generated in the presence of coatomer (arrowheads). Bars, 5 µm.